Glansbeek H L, van Beuningen H M, Vitters E L, van der Kraan P M, van den Berg W B
Department of Rheumatology, University Hospital Nijmegen, The Netherlands.
Protein Expr Purif. 1998 Mar;12(2):201-7. doi: 10.1006/prep.1997.0819.
Transforming growth factor-beta (TGF-beta) is a potent regulator of cell metabolism, proliferation, and differentiation. To study the role of endogenous TGF-beta in processes such as tissue repair and inflammation, potent and specific inhibitors are required. Because the type II TGF-beta receptor (TGF beta RII) has a high affinity for TGF-beta, the extracellular domain of TGF beta RII (TGF-beta sRII) was expressed in Pichia pastoris and Escherichia coli. Expression of the soluble TGF beta sRII using P. pastoris resulted in a soluble, heterogeneously glycosylated protein which was secreted into the medium. Although expression of TGF beta sRII in E. coli resulted in the formation of insoluble inclusion bodies, solubilization and refolding resulted in a biologically active protein. Because in both systems a C-terminal 6x His coding sequence was inserted behind the coding sequence for the extracellular domain of TGF beta RII the recombinant proteins could be purified by a powerful, single-step procedure using a Ni-NTA agarose. The purified proteins appeared to be potent inhibitors of TGF-beta 1 and TGF-beta 3. In contrast, TGF beta sRII was less effective in neutralization of TGF-beta 2. In conclusion, biologically active TGF beta sRII can be produced using P. pastoris and E. coli expression systems. The ease of these expression systems, the powerful single step purification and low costs makes it possible to produce TGF beta s RII in large amounts to further elucidate the role of TGF-beta 1 and TGF-beta 3 in physiological processes like tissue repair and inflammation.
转化生长因子-β（TGF-β）是细胞代谢、增殖和分化的有效调节因子。为了研究内源性TGF-β在组织修复和炎症等过程中的作用，需要强效且特异性的抑制剂。由于II型TGF-β受体（TGFβRII）对TGF-β具有高亲和力，因此TGFβRII的细胞外结构域（TGF-βsRII）在巴斯德毕赤酵母和大肠杆菌中进行了表达。使用巴斯德毕赤酵母表达可溶性TGF-βsRII产生了一种可溶的、糖基化不均一的蛋白质，该蛋白质分泌到培养基中。尽管在大肠杆菌中表达TGF-βsRII导致形成不溶性包涵体，但溶解和重折叠产生了一种具有生物活性的蛋白质。由于在这两个系统中，一个C末端6x His编码序列插入到TGF-βRII细胞外结构域的编码序列之后，重组蛋白可以通过使用Ni-NTA琼脂糖的强大单步程序进行纯化。纯化后的蛋白质似乎是TGF-β1和TGF-β3的有效抑制剂。相比之下，TGF-βsRII在中和TGF-β2方面效果较差。总之，使用巴斯德毕赤酵母和大肠杆菌表达系统可以产生具有生物活性的TGF-βsRII。这些表达系统的简便性、强大的单步纯化和低成本使得大量生产TGF-βsRII成为可能，以进一步阐明TGF-β1和TGF-β3在组织修复和炎症等生理过程中的作用。
